Analysis of tissue cadmium distribution in chronic cadmium-exposed mice using in-air micro-PIXE

This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE) and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with 3 mg/kg body weight of CdCl₂ thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity. The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced toxicity.     

Influence of dietary iron deficiency on acute metal intoxication

The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal and intestinal metallothionein (MT), disposition of the metals, and alterations in hematological parameters was investigated in rats. The administration of cadmium induced the hepatic, renal and intestinal MT while that of nickel or lead induced hepatic MT only. However, dietary iron deficiency did not influence the cadmium induced tissue MT but enhanced the ability of nickel or lead to restore the normal synthesis of renal and intestinal MT lowered under the influence of reduced body iron status. The accumulation of lead in liver and kidney and that of cadmium enhanced in liver only, while tissue deposition of nickel remained unaffected by iron deficiency. The induction of hepatic MT by three metals appears related to the concomitant rise in the hepatic zinc, calcium and iron levels in normal rats. However, dietary iron deficiency increased the hepatic zinc in response to nickel or cadmium and that of heptic calcium in response to lead.     

Discriminative uptake of metals by the liver and its relation to induction of metallothionein by cadmium, copper and zinc

1. Disappearance from plasma and uptake by the liver of cadmium (Cd), copper (Cu) and zinc (Zn) were examined with a view to studying the biological discrimination between essential and non-essential heavy metals. 2. Cd injected intravenously at a single dose of 0.8 mg/kg body wt disappeared from rat plasma rapidly within about 10 min, while Cu and Zn injected at the same dose disappeared slowly in plasma and decreased to the control level after about 3 hr. 3. Uptake of Cd by the liver corresponded well with the rapid disappearance from plasma, while Cu and Zn accumulated slowly in the liver and their concentrations started to increase after their plasma concentrations had decreased. 4. Metallothionein was induced in the liver at a similar time course for the three metals, suggesting the presence of discriminative uptake processes by the liver with similar or the same detoxification mechanisms through induction of metallothionein.     

Dietary cadmium decreases lipid peroxidation in the liver and kidneys of the bank vole (Clethrionomys glareolus).

The effect of elevated levels of dietary cadmium on lipid peroxidation in the liver and kidneys of a small rodent, the bank vole, was determined in the present study. Males and females, aged 1 month, were given diets containing 0.40 and 80 mg Cd per kg; liver and kidneys were removed for TBA-RS as well as iron, copper, zinc, cadmium and metallothionein analyses at the end of 6 weeks. Dietary Cd significantly decreased the TBA-RS level in the liver and kidneys of both sexes; however, this effect appeared to be dose-dependent only for the male liver. The changes in hepatic and renal TBA-RS paralleled closely those of tissue iron. Copper concentration decreased significantly only in the male liver, while hepatic and renal zinc were not influenced by dietary Cd. The concentrations of Cd and metallothionein in the liver and kidneys increased significantly in a dose-dependent fashion. Regression analysis confirmed that TBA-RS in both organs correlated closely with iron. The data suggest that dietary Cd decreases hepatic and renal lipid peroxidation indirectly, through lowering the tissue iron concentration.     

Excretion of urinary cadmium, copper, and zinc in cadmium-exposed and nonexposed subjects, with special reference to urinary excretion of β2-microglobulin and metallothioneinand metallothionein

The objectives of this study were to examine the association between urinary excretion of cadmium (U-Cd), copper (U-Cu), and zinc (U-Zn) and the severity of two different indicators of renal toxicity (urinary excretion of beta2-microglobulin [U-beta2-MG] and metallothionein [U-MT]) in Cd-exposed subjects compared to controls, and to assess the physiologic mechanisms by which the exposure to environmental Cd affects U-Cd, U-Cu, and U-Zn. The target population included 3508 Cd-exposed and 294 nonexposed participants who received a health survey conducted among the population of the Kakehashi River basin. Increases of U-Cd, U-beta2-MG, and U-MT in the Cd-exposed population were observed relative to excretion of these substances in controls. Regression analysis using a general linear model revealed that the correlations between U-Cd or U-Cu, and U-beta2-MG and between U-Cd, U-Cu or U-Zn, and U-MT were statistically significant in both sexes, but the correlation between U-Zn and U-beta2-MG excretion was significant only in men. These results suggest U-Cd and U-Cu is affected by dysfunction in renal tubular absorption (indicated by U-beta2-MG), whereas not only U-Cd and U-Cu but also U-Zn appear to be a function of renal cellular desquamation (indicated by U-MT).     

Properties of metallothionein induced by zinc, copper and cadmium in the frog, Xenopus laevis

1. Repeated injections of zinc (Zn) and copper (Cu) into the frog Xenopus laevis caused accumulations of the respective metals in the liver and kidney. 2. The accumulated metals in the liver supernatant fractions were present as Zn- and Cu-binding proteins of the same properties as that of metallothionein (MT) induced by cadmium (Cd) injections. 3. The affinity of Zn, Cu and Cd ions to the metal-binding protein was in the decreasing order of Cu, Cd and Zn. 4. The Xenopus MT induced by Cd was unstable and disrupted easily to give two peaks as if the MT consists of two isometallothioneins.     

Zinc, antioxidant systems and metallothionein in metal mediated-apoptosis: biochemical and cytochemical aspects

Copper, zinc and iron are essential metals for different physiological functions, even though their excess can lead to biological damage. This review provides a background of toxicity related to copper, iron and zinc excess, biological mechanisms of their homeostasis and their respective roles in the apoptotic process. The antioxidant action of metallothionein has been highlighted by summarizing the most important findings that confirm the role of zinc in cellular protection in relation to metallothionein expression and apoptotic processes. In particular, we show that a complex and efficient antioxidant system, the induction of metallothionein and the direct action of zinc have protective roles against oxidative damage and the resulting apoptosis induced by metals with redox proprieties. In addition, to emphasize the protective effects of Zn and Zn-MT in Cu and Fe-mediated oxidative stress-dependent apoptosis, some aspects of apoptotic cell death are shown. The most widely used cytochemical techniques also have been examined in order to critically evaluate the available data from a methodological point of view. The observations on the role of Zn and MT could potentially develop new applications for this metal and MT in biomedical research.     

Influence of dietary cadmium on the distribution of the essential metals copper,zinc and iron in tissues of the rat

The effect of long-term dietary cadmium treatment upon the distribution of the metals copper, iron and zinc has been compared in various organs of male and female rats. The renal accumulation of cadmium was similar in both sexes without a plateau being reached. In contrast, the hepatic accumulation of cadmium was higher in the female than in the male rat and a plateau was observed after 30–35 weeks of dietary cadmium treatment. Most of the cadmium which accumulated in these organs was recovered in the metallothionein fraction and the concentration of hepatic cadmiumthionein in the female rat was correspondingly higher than in the male rat. Accumulation of cadmium was associated with an increased zinc concentration in the liver and an increased copper concentration in the kidney; these increases were correlated with increases in liver and kidney metallothioneins induced by cadmium. Uptake of cadmium into organs other than liver and kidney occurred to a small extent but was not associated with changes in the concentration of copper and zinc. Cadmium also accumulated in the intestinal mucosa where it could be recovered in a fraction corresponding to metallothionein. A loss of iron from the liver and kidney was also observed following dietary cadmium treatment and involved mainly a loss of iron from ferritin.     

Metal composition of human hepatic and renal metallothionein

This article is based on data on the levels of metals (Cd, Zn, Cu) and metallothionein (MT) determined radiochemically with²⁰³Hg in renal cortex and liver of 137 autopsy cases. From this number, for 23 cases, the gel filtration of the cytoplasmic fraction of the organs was performed. The molar content of metals in the MT fraction (Sephadex G-50) amounted to 46.9, 50.2, and 2.0% for Cd, Zn, and Cu in renal cortex, respectively, and to 8.3, 83.6, and 9.1% for Cd, Zn, and Cu in the liver, respectively. In parallel with the increase of Cd and MT in renal cortex, increasing saturation was found of the MT fraction by Cd, occurring at the expense of Zn and Cu. Equimolar amounts of Cd and Zn in the MT fraction are found at Cd level of 0.5 μmol Cd/g wet wt of renal cortex. In the liver, analogous dependency (elevation of %Zn, depression of %Cd and %Cu) were observed in relation to Zn and MT levels in this organ. The basic level of Zn (not bound with MT) was estimated at 0.5 μmol/g for both renal cortex and liver. A deficit of non-MT Zn in kidneys is proposed as an alternative mechanism of toxic Cd action.     

Effect of 6-mercaptopurine on mineral and metallothionein metabolism in the mouse

The effect of the anticancer drug 6-mercaptopurine (6-MP) on mineral metabolism was investigated in mice. C57Bl/6J female mice were injected intraperitoneally with 6-MP at 100 mg/kg body wt for one, two, four, or five consecutive days. On d 6 of the study, liver, kidney, and intestine were removed, and concentrations of zinc, copper, iron, manganese, magnesium, and calcium were measured. Hepatic concentrations of zinc, copper, iron, and calcium became higher as the number of drug injections increased. To determine if the altered mineral metabolism was a function of a drug-induced, acute-phase response, liver metallothionein and plasma ceruloplasmin were measured. Metallothionein concentrations in the liver became higher with increased number of injections, correlating with the stepwise increase in hepatic zinc. Gel filtration chromatography showed that most of the increase in liver zinc concentration was associated with a protein of mol wt of 6000–8000, the approximate weight of metallothionein. Ceruloplasmin concentrations were not affected by 6-MP injection. These results suggested that 6-MP alters zinc metabolism by sequestering zinc into the liver via induction of metallothionein synthesis and that the drug may induce an acute-phase response with an atypical acute-phase protein profile.     

Role of thiocyanate ion in metallothionein induction and in endogenous distribution of essential elements in the rat liver

Thiocyanate is the major toxic metabolite of hydrogen cyanide, a toxic substance the organism may be exposed to as a result of cigarette smoking or industrial pollution. The complex interactions existing between metals and metallothionein induction are well known. However, the possible role of thiocyanate, which is also an anion, has not been established yet. Considering the interactions between metals and the metallothioneins, in this study the relationship between thiocyanate and the in vivo distribution of hepatic metallothionein and zinc, copper, iron, calcium, magnesium, and manganese are investigated in rats. This study implies that thiocyanate has, to some extent, an effect on the in vivo expression of metallothionein and endogenous distribution of essential elements in rat liver. Elevated levels of metallothionein and changes in hepatic concentrations of essential elements have suggested a role for thiocyanate in cellular metabolism and it might reflect a direct role of thiocyanate on alteration of cellular functional activities.     

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn²⁺ uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of {–}SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl₂ at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd²⁺, Zn²⁺ and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 μg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V_max) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.     

Hepatic and renal metallothionein induction in the rat and mouse after treatment with furosemide.

Induction of hepatic and and renal metallothionein by furosemide was studied in the rat and mouse. Treatment of mice with 200 and 300 mg/kg furosemide elevated hepatic metallothionein by 117% and 366%, while renal metallothionein was induced by 29% and 380%, respectively. In the rat the drug was less potent i.e. liver metallothionein was increased by 167% and 217% following injection of 300 and 400 mg/kg furosemide, respectively, whereas kidney was not significantly changed by this treatment. The mouse hepatic and renal metallothionein was identified as zinc-containing thionein by Sephadex G-75 gel filtration (Ve/Vo = 2.0). In both species maximal induction was observed 24 hours post exposure. However, the mouse hepatic and renal metallothionein content declined after additional 24 hours whereas the rat metalloprotein was not reduced even after 72 hours of treatment. It is suggested that alterations in metal homeostasis may be responsible for hepatic and renal metallothionein induction caused by furosemide.     

Male sterility of transgenic mice carrying exogenous mouse interferon-beta gene under the control of the metallothionein enhancer-promoter.

As an approach to elucidating the roles of interferon (IFN) in the normal physiology and diseases of animals, transgenic mice carrying extra mouse IFN-beta genes under the control of a mouse metallothionein I enhancer-promoter were constructed. Upon induction with Cd2+, IFN activity (15-430 IU/ml) was detected in the sera of six out of ten transgenic mouse lines so far obtained. Synthesis of mRNA of the transgene was observed in the liver, the testis and less abundantly in the brain. Interestingly, IFN mRNA was constitutively synthesized in the testis where substantial levels of IFN accumulated without heavy metal induction, whereas synthesis in the liver was mostly dependent on induction by CD2+. Since IFN activity in the serum also depended on heavy metal induction, the IFN in the serum may be produced mainly in the liver. All males expressing the IFN gene in the testis were found to be sterile. Testes were involuted and contained few mature sperm, and degeneration of spermatocytes and spermatids was observed. These findings suggest that high levels of IFN are harmful to spermatogenesis and can cause male sterility.     

Studies on the role of iron in the reversal of cadmium toxicity in chicks

Studies were conducted to determine the effect of dietary iron (Fe) levels ranging from a deficiency to an excess on the toxicity of cadmium (Cd) in chicks. In Fe-deficient animals, cadmium was found to be more toxic than in Fe supplemented animals as measured by growth. The liver Cd burdens were increased significantly in the presence of dietary Fe supplementation, and there was a significant Cd−Fe interaction in the Cd concentration of the kidney, indicating that iron deficiency increased the concentration of Cd in the kidneys of those chicks receiving this element. Cd tended to reduce the Fe concentration in both the liver and kidney. The absorption of Cd as measured by the amount of¹⁰⁹Cd that disappeared from an isolated duodenal segment in one h was not affected by the Fe content of the diet, but the amount of isotope appearing in the liver compared to the amount present in the blood was increased in the Fe supplemented chicks. Separation of the Cd binding ligands by column chromatography revealed that more of the Cd in the liver, but not the kidney, was associated with ligands which eluted in a column volume that contained metallothionein in those chicks receiving Fe than in the livers from Fe deficient animals. The inverse relationship between the amount of Cd bound to the metallothionein containing fraction and toxicity may be related causally. Paper No. 10538 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names in this publication does not imply endorsement by the NC Agricultural Research Service of the products named nor criticism of similar ones not mentioned.     

不同因素诱导豚鼠肝中金属硫蛋白的研究

通过皮下注射的方法诱导豚鼠产生金属硫蛋白（ＭＴ）,研究了重金属元素（Ｃｄ）、微量元素（Ｃｕ,Ｚｎ）及有机试剂（ＣＣｌ４,在体内可产生自由基）等因素的诱导与豚鼠肝脏中ＭＴ不同亚型的含量及金属结合状态的变化关系．实验结果表明,微量元素及有机试剂的诱导可使豚鼠肝脏中ＭＴ１的产量明显高于ＭＴ２,说明在体内ＭＴ１在参与微量元素的储存及清除自由基功能方面比ＭＴ２强．在重金属元素诱导下体内ＭＴ１对重金属元素的结合量远远大于ＭＴ２．表明ＭＴ１的重金属解毒能力比ＭＴ２强．上述实验结果与对不同亚型ＭＴ生物学功能差异的体外研究结果相吻合．此外,无论采用上述何种因素诱导,所得ＭＴ中均结合有Ｃｕ．对Ｃｕ在ＭＴ形成过程中的作用也进行了初步探讨．     

Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

Cd/Zn exposure interactions on metallothionein response in Eisenia fetida (Annelida, Oligochaeta)

We studied metallothionein (MT) response in the manure worm Eisenia fetida after exposures to cadmium (Cd), zinc (Zn) or cadmium and zinc spiked media. MT was studied both at the protein level by Dot Immunobinding Assay, (DIA) and at the expression level by Northern blotting. Cd was highly accumulated by worms whereas Zn body concentration was regulated. In addition, Zn would limit Cd accumulation in worms exposed to low Cd concentrations (1 and 8 mg Cd kg(-1) of dry soil). Exposure to a mixture of Cd and Zn at high concentrations increased cytosolic MT levels. This increase would allow worms to regulate body Zn concentrations and also to limit Cd toxicity. Cd exposures increased gene expression of Cd-binding MT isoform (MT 2A) whereas Zn did not. However, when both metals were at high concentrations in the exposure medium, this expression was further increased. Several hypotheses are proposed to explain the results and the best approach to estimate metal exposure of this earthworm species is given. Further experiments have now to be performed to evaluate the usefulness of these MT responses for field contaminated soils toxicity assessment using this earthworm species.     

Effects of ingestion of cadmium-polluted rice or low-dose cadmium-supplemented diet on the endogenous metal balance in female rats

The concentrations of endogenous metal ions in liver, kidney, and bone tissues of female rats were measured after ingestion of cadmium-polluted rice (1.24 ppm as Cd) or cadmium-supplemented rice (1.24 and 4.96 ppm) for 2 or 4 mo. The metal accumulated mainly in the kidneys and in the liver. The concentration of cadmium (Cd) in the kidneys of rats fed a 1.24-ppm Cd-supplemented diet was significantly higher than in the Cd-polluted rice group. After 2 mo, the levels of iron and sodium in the liver were elevated in the Cd-polluted rice group, but not in the 1.24-ppm Cd-supplemented group, as compared to controls. The zinc concentration in the Cd-polluted rice group was decreased. The concentration of copper in the kidneys was increased for all Cd-containing diet groups. After 4 mo, the effects of Cd on essential metals in the Cd-polluted and 1.24-ppm Cd-supplemented groups had almost disappeared, although several metal ions in selected organs in the 4.96-ppm Cd-supplemented group showed more prominent changes than in the group exposed for 2 mo. These results suggest that the effects of short-term exposure to Cd on essential metal balance are stronger for rice-bound Cd than for inorganic Cd, although the absorption rate of Cd in Cd-polluted rice may be lower than that of cadmium chloride added to rice.